RESUMEN
An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Proteínas Bacterianas/metabolismo , Eritrocitos/inmunología , Bacilos y Cocos Aerobios Gramnegativos/enzimología , Hexosaminidasas/metabolismo , Secuencia de Carbohidratos , Hexosaminidasas/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , alfa-N-AcetilgalactosaminidasaRESUMEN
An alpha-galactosidase that inactivates the group specificity of B erythrocytes (group III) of human blood and does not affect A erythrocytes (group II) was isolated from the marine bacterium Pseudoalteromonas sp. KMM 701. The enzyme preparation did not contain lectin, hemolytic, sialidase, endoglycanase, or glycosidase activities. The enzyme is stable at 20 degreesC for 24 h, has pH optimum for catalysis within the range of 6.7-7.7, and is stable to high concentrations of NaCl. It is 4-fold more efficient than the alpha-galactosidase from green coffee beans. At pH 7.0 the Km for p-nitrophenyl-alpha-D-galactopyranoside is 0.29 mM. The molecular weight of the enzyme determined by gel-filtration is 195 +/- 5 kD. The alpha-galactosidase is denatured by urea and guanidine hydrochloride. Its activity does not depend on the presence of metal ions. It contains a sulfhydryl group essential for its catalytic activity.
Asunto(s)
Bacterias Aerobias Gramnegativas/enzimología , alfa-Galactosidasa/aislamiento & purificación , Sistema del Grupo Sanguíneo ABO/química , Secuencia de Carbohidratos , Estabilidad de Enzimas , Eritrocitos/química , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Peso Molecular , Agua de Mar/microbiología , Especificidad por Sustrato , alfa-Galactosidasa/química , alfa-Galactosidasa/metabolismoRESUMEN
A comparative analysis of carbohydrate chains from human normal IgG and myeloma IgG (subclass 4) was carried out using HPLC. It is shown that both IgGs contain the same carbohydrate chains (biantennary and bisected) but the relative amount of bisected and incomplete chains (with fewer terminal galactose residues) is higher in myeloma IgG4. Two earlier unknown minor oligosaccharides were isolated from normal IgG and structurally studied by means of the partial hydrolysis and the Smith degradation.
Asunto(s)
Carbohidratos/análisis , Inmunoglobulina G/análisis , Proteínas de Mieloma/análisis , Cromatografía Líquida de Alta Presión , Humanos , Oligosacáridos/análisisRESUMEN
A method for specific fragmentation of the polypeptide backbone of glycoproteins at the glycosylated serine and threonine residues has been developed. The fragmentation includes beta-elimination of the carbohydrate chains, followed by bromination of the resulting enamine groups, and cleavage of the brominated amino acid residues by alkaline sodium borohydride. The method was employed for fragmentation of the peptide core of pig blood-group substance H. Essentially all the serine and threonine residues were shown to be O-glycosylated, and rather frequently either adjacent or separated by a single amino acid (mainly alanine). When they were separated by two or three amino acid residues, proline was preponderant.